Plague is a zoonosis that is present in wild rodent populations worldwide and is transmitted primarily by fleas. Yersinia pestis, the plague bacillus, is unique among the enteric group of gram-negative bacteria in having adopted an arthropod-borne route of transmission. Y. pestis has evolved in such a way as to be transmitted during the brief encounter between a feeding flea and a host. A transmissible infection primarily depends on the ability of Y. pestis to grow in the flea as a biofilm that is embedded in a complex extracellular matrix. Bacteria in the biofilm phenotype are deposited into the dermis together with flea saliva, elements which cannot be satisfactorily mimicked by needle-injection of Y. pestis from laboratory cultures. The objective of this project is to identify and determine the function of Y. pestis genes that mediate flea-borne transmission and the initial encounter with the host innate immune system at the infection site in the skin. We study the interaction of Y. pestis with its insect vector by using an artificial feeding apparatus to infect fleas with uniform doses of wild-type or specific Y. pestis mutants. We seek to identify Y. pestis genes that are required for the bacteria to infect the flea midgut and to produce a biofilm that blocks the flea foregut and that is required for efficient transmission. The strategy entails first identifying bacterial genes that are differentially expressed in the flea by gene expression analysis and other techniques. Specific mutations are then introduced into these genes, and the mutants tested for their ability to infect and block the flea vector. Identification of such transmission factors allows further studies into the molecular mechanisms of the bacterial infection of the flea vector. Detailed understanding of the interaction with the insect host may lead to novel strategies to interrupt the transmission cycle. Our studies of flea vector competence and vectorial capacity will be useful to develop more realistic mathematical modeling of the epidemiology of plague transmission and the conditions that lead to plague epizootics. During FY2015, we continued to investigate the regulation of Y. pestis genes required for the biofilm life stage in the flea vector. We previously reported that a major bacterial gene regulatory system (known as PhoP-PhoQ) is induced during infection of the flea vector and is required to produce a normal transmissible infection. Because PhoP-PhoQ upregulates several genes required for bacterial resistance to innate immunity, induction of the PhoP-PhoQ system in the arthropod vector prior to transmission may preadapt Y. pestis to resist the initial encounter with the mammalian innate immune response. This year, we published the results of microarray experiments that compared the in vitro and in vivo (i.e., in the flea) transcriptomes of wild-type and phoP-negative Y. pestis. The analysis indicated that the PhoPQ signal transduction-gene regulatory system is induced by low pH in the flea gut, and that PhoP modulates gene expression leading to physiological adaptation to acid and other stresses encountered during infection of the flea. We also continue to investigate the genetic changes that led to the evolutionarily recent transition of Y. pestis to an arthropod-borne transmission route. Y. pseudotuberculosis, the recent ancestor of Y. pestis, causes acute toxicity and 30-40% mortality to fleas that ingest it in a blood meal. In contrast, Y. pestis is not orally toxic to fleas, indicating that an ancestral insecticidal toxin was lost, and that this was important in the recent adaption to the flea-borne transmission route. In FY2015 we completed a project that identified the Yersinia urease enzyme as the toxic factor. All Y. pestis are urease-negative because ureD is a pseudogene; our work indicates that loss of urease activity was positively selected during the evolution of Y. pestis because it increased flea-borne transmission potential. This provided another specific example of how gene loss appears to have played a significant role in Y. pestis evolution. In collaboration with the Genomics Unit of the RML Research Technologies Branch, we are examining and characterizing the transcriptomes of Y. pestis, Y. pseudotuberculosis, and our biofilm-producing Y. pseudotuberculosis mutant during growth in vitro and during infection of the flea, with the goal of identifying genes and gene regulatory pathways that are important for flea-borne transmission. The in vivo and in vitro transcriptomic comparisons between these three strains are designed to broadly identify candidate components of biofilm regulatory pathways and other genes important for the recent evolutionary adaptation to flea-borne transmission. Significant differences in the expression of orthologous genes in the flea might be indicative of evolutionary changes in gene regulatory pathways. This year we have completed a study on the vector competence of the cat flea Ctenocephalides felis. We used a new experimental system to quantitate the CFUs transmitted by cohorts of fleas as well as individual fleas over a four-week period following a standardized infectious blood meal. This allowed us to compare the relative efficiency of two mechanisms by which fleas transmit Y. pestis: biofilm-independent early-phase transmission during the first few days after the infectious blood meal and proventricular biofilm-dependent transmission, which occurs from 1-4 weeks after the infectious blood meal. These data will improve mathematical models used to assess the relative importance of the two transmission mechanisms in plague ecology. These results will be published in FY2016.